Transplantation of aged LSKs into young mice reverses the aged HPC mitochondrial phenotype. (A) Schematic of the experiment for the adoptive transfer of young and aged LSKs into young mice. BM was isolated from young and aged C57Bl/6 mice (CD45.2⁺), and LSKs were separated by FACS. They were transplanted into young PepCboy mice (CD45.1⁺). At 12 weeks, mice were killed, their BM was isolated, and terminal blood samples were taken. Flow cytometry was used to assess the engraftment. (B) Percentage of CD45.2⁺ engrafted LSKs, LS-Ks, MPPs, and PB cells in mice transplanted with LSKs from either young or aged mice. (C) Analysis of PB lineages was performed to evaluate the proportion of lymphoid (CD3⁺ B220⁺) and myeloid (Gr1⁺ CD11b⁺) cells in mice transplanted with LSKs from young or aged mice. (D) CD45.2⁺ LSK cell counts per 100 000 BM cells in mice transplanted with LSKs from young or aged mice. (E) Frequency of CD45.2⁺ TMRM^hi LSKs, LS-Ks, and MPPs isolated from mice 12 weeks after transplant with young or aged LSKs. (F) Schematic of the experiment. After engraftment of LSKs from aged C57Bl/6 mice was confirmed in the young PepCboy recipients at 12 weeks, the transplanted PepCboy mice were treated with 0.5 mg/kg LPS or vehicle control. After 16 hours, their BM was isolated, and analysis was conducted on CD45.2⁺ engrafted cells. (G) Number of LSKs in mice transplanted with aged LSKs and then treated with LPS or vehicle control. (H) Frequency of TMRM^hi LSKs, LS-Ks, and MPPs from control and LPS-treated mice. (I) Basal and maximal respiration of LKs after LPS treatment compared with controls, measured by Seahorse metabolix flux analysis. (J) Changes in OCR compared with extracellular acidification rate (ECAR) in control and LPS-treated mice previously transplanted with LSKs from aged mice. ∗P < .05, ∗∗P < .01, ∗∗∗∗P < .0001 using the Mann-Whitney U test or two-way analysis of variance. Con, vehicle control; ns, not significant.